This invention relates to the field of immunoglobulin production and to modification of naturally occurring immunoglobulin amino acid sequences. Specifically, the invention relates to using recombinant techniques to produce both immunoglobulins which are analogous to those normally found in vertebrate systems and to take advantage of these gene modification techniques to construct chimeric or other modified forms.
A. Immunoglobulins and Antibodies
Antibodies are specific immunoglobulin polypeptides produced by the vertebrate immune system in response to challenge by foreign proteins, glycoproteins, cells, or other antigenic foreign substances. The sequence of events which permits the organism to overcome invasion by foreign cells or to rid the system of foreign substances is at least partially understood. An important part of this process is the manufacture of antibodies which bind specifically to a particular foreign substance. The binding specificity of such polypeptides to a particular antigen is highly refined, and the multitude of specificities capable of being generated by the individual vertebrate is remarkable in its complexity and variability. Thousands of antigens are capable of eliciting responses, each almost exclusively directed to the particular antigen which elicited it.
Immunoglobulins include both antibodies, as above described, and analogous protein substances which lack antigen specificity. The latter are produced at low levels by the lymph system and in increased levels by myelomas.
A.1 Source and Utility
Two major sources of vertebrate antibodies are presently utilized--generation in situ by the mammalian B lymphocytes and in cell culture by B-cell hybrids. Antibodies are made in situ as a result of the differentiation of immature B lymphocytes into plasma cells, which occurs in response to stimulation by specific antigens. In the undifferentiated B cell, the portions of DNA coding for the various regions on the immunoglobulin chains are separated in the genomic DNA. The sequences are reassembled sequentially prior to transcription. A review of this process has been given by Gough, Trends in Biochem Sci, 6: 203 (1981). The resulting rearranged genome is capable of expression in the mature B lymphocyte to produce the desired antibody. Even when only a single antigen is introduced into the sphere of the immune system for a particular mammal, however, a uniform population of antibodies does not result. The in situ immune response to any particular antigen is defined by the mosaic of responses to the various determinants which are present on the antigen. Each subset of homologous antibody is contributed by a single population of B-cells--hence in situ generation of antibodies is "polyclonal".
This limited but inherent heterogeneity has been overcome in numerous particular cases by use of hybridoma technology to create "monoclonal" antibodies (Kohler, et al., Eur. J. Immunol., 6: 511 (1976)). In this process, splenocytes or lymphocytes from a mammal which has been injected with antigen are fused with a tumor cell line, thus producing hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell. The hybrids thus formed we segregated into single genetic strains by selection, dilution, and regrowth, and each strain thus represents a single genetic line. They therefore produce immunoreactive antibodies against a desired antigen which are assured to be homogenous, and which antibodies, referencing their pure genetic parentage, are called "monoclonal". Hybridoma technology has to this time been focused largely on the fusion of murine lines, but human-human hybridomas (Olsson, L. et al., Proc. Natl. Acad. Sci. (USA), 77: 5429 (1980)); human-murine hybridomas (Schlom, J. , et al. (ibid) 77: 6841 (1980)) and several other xenogenic hybrid combinations have been prepared as well. Alternatively, primary, antibody producing, B cells have been immortalized in vitro by transformation with viral DNA.
Polyclonal, or, much more preferably monoclonal, antibodies have a variety of useful properties similar to those of the present invention. For example, they can be used as specific immunoprecipitating reagents to detect the presence of the antigen which elicited the initial processing of the B cell genome by coupling this antigen-antibody reaction with suitable detection techniques such as labeling with radioisotopes or with enzymes capable of assay (RIA, EMIT, and ELISA). Antibodies are thus the foundation of immuno diagnostic tests for many antigenic substances. In another important use, antibodies can be directly injected into subjects suffering from an attack by a substance or organism containing the antigen in question to combat this attack. This process is currently in its experimental stages, but its potential is clearly seen. Third, whole body diagnosis and treatment is made possible because injected antibodies are directed to specific target disease tissues, and thus can be used either to determine the presence of the disease by carrying with them a suitable label, or to attack the diseased tissue by carrying a suitable drug.
Monoclonal antibodies produced by hybridomas, while theoretically effective as suggested above and clearly preferable to polyclonal antibodies because of their specificity, suffer from certain disadvantages. First, they tend to be contaminated with other proteins and cellular materials of hybridoma, (and, therefore, mammalian) origin. These cells contain additional materials, notably nucleic acid fragments, but protein fragments as well, which are capable of enhancing, causing, or mediating carcinogic responses. Second, hybridoma lines producing monoclonal antibodies tend to be unstable and may alter the structure of antibody produced or stop producing antibody altogether (Kohler, G., et al. Proc. Natl. Acad. Sci (USA) 77: 2197 (1980); Morrison, S. L., J. Immunol. 123: 793 (1979)). The cell line genome appears to alter itself in response to stimuli whose nature is not currently known, and this alteration may result in production of incorrect sequences. Third, both hybridoma and B cells inevitably produce certain antibodies in glycosylated form (Melchers, F., Biochemistry, 10: 653 (1971)) which, under some circumstances, may be undesirable. Fourth, production of both monoclonal and polyclonal antibodies is relatively expensive. Fifth, and perhaps most important, production by current techniques (either by hybridoma or by B cell response) does not permit manipulation of the genome so as to produce antibodies with more effective design components than those normally elicited in response to antigens from the mature B cell in situ. The antibodies of the present invention do not suffer from the foregoing drawbacks, and, furthermore, offer the opportunity to provide molecules of superior design.
Even those immunoglobulins which lack the specificity of antibodies are useful, although over a smaller spectrum of potential uses than the antibodies themselves. In presently understood applications, such immunoglobulins are helpful in proteins replacement therapy for globulin related anemia. In this context, an inability to bind to antigen is in fact helpful, as the therapeutic value of these proteins would be impaired by such functionality. At present, such non-specific antibodies are derivable in quantity only from myeloma cell cultures suitably induced. The present invention offers an alternative, more economical source. It also offers the opportunity of canceling out specificity by manipulating the four chains of the tetramer separately.
A.2 General Structure Characteristics
The basic immunoglobin structural unit in vertebrate systems is now well understood (Edelman, G. M., Ann. N.Y. Acad. Sci., 190: 5 (1971)). The units are composed of two identical light polypeptide chains of molecular weight approximately 23,000 daltons, and two identical heavy chains of molecular weight 53,000-70,000. The four chains are joined by disulfide bonds in a "Y" configuration wherein the light chains bracket-the heavy-chains starting at the mouth of the Y and continuing through the divergent region as shown in FIG. 1. The "branch" portion, as there indicated, is designated the Fab region. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, with some subclasses among them, and the nature of this chain, as it has a long constant region, determines the "class"of the antibody as IgG, IgM, IgA, IgD, or IgE. Light chains are classified-as either kappa or lambda. Each heavy chain class can be prepared with either kappa or lambda light chain. The light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages when the immunoglobulins are generated either by hybridomas or by B cells. However, if non-covalent association of the chains can be effected in the correct geometry, the aggregate will still be capable of reaction with antigen, or of utility as a protein supplement as a non-specific immunoglobulin.
The amino acid sequence runs from the N-terminal end at the top of the Y to the C-terminal end at the bottom of each chain. At the N-terminal end is a variable region which is specific for the antigen which elicited it, and is approximately 100 amino acids in length, there being slight variations between light and heavy chain and from antibody to antibody. The variable region is linked in each chain to a constant region which extends the remaining length of the chain. Linkage is seen, at the genomic level, as occurring through a linking sequence known currently as the "J" region in the light chain gene, which encodes about 12 amino acids, and as a combination of "D" region and "J" region in the heavy chain gene, which together encode approximately 25 amino acids.
The remaining portions of the chain are referred to as constant regions and within a particular class do not to vary with the specificity of the antibody (i.e., the antigen eliciting it),
As stated above, there are five known major classes of constant regions which determine the class of the immunoglobulin molecule (IgG, IgM, IgA, IgD, and IgE corresponding to .gamma., .mu., .alpha., .delta., and .epsilon. heavy chain constant regions). The constant region or class determines subsequent effector function of the antibody, including activation of complement (Kabat, E. A., Structural Concepts in Immunology and Immunochemistry, 2nd Ed., p. 413-436, Holt, Rinehart Winston (1976)), and other cellular responses (Andrews, D. W., et al., Clinical Immunobiology pp 1-18, W. B. Sanders (1980); Kohl, S., et al., Immunology, 48: 187 (1983)); while the variable region determines the antigen with which it will react.
B. Recombinant DNA Technology
Recombinant DNA technology has reached sufficient sophistication that it includes a repertoire of techniques for cloning and expression of gene sequences. Various DNA sequences can be recombined with some facility, creating new DNA entities capable of producing heterologous protein product in transformed microbes and cell cultures. The general means and methods for the in vitro ligation of various blunt ended or "sticky" ended fragments of DNA, for producing expression vectors, and for transforming organisms are now in hand.
DNA recombination of the essential elements (i.e., an origin of replication, one or more phenotypic selection characteristics, expression control sequence, heterologous gene insert and remainder vector) generally is performed outside the host cell. The resulting recombinant replicable expression vector, or plasmid, is introduced into cells by transformation and large quantities of the recombinant vehicle is obtained by growing the transformants. Where the gene is properly inserted with reference to portions which govern the transcription and translation of the encoded DNA message, the resulting expression vector is useful to produce the polypeptide sequence for which the inserted gene codes, a process referred to as "expression." The resulting product may be obtained by lysis, if necessary, of the host cell and recovery of the product by appropriate purifications from other proteins.
In practice, the use of recombinant DNA technology can express entirely heterologous polypeptides--so-called direct expression--or alternatively may express a heterologous polypeptide fused to a portion of the amino acid sequence of a homologous polypeptide. In the latter cases, the intended bioactive product is sometimes rendered bioinactive within the fused, homologous/heterologous polypeptide until it is cleaved in an extracellular environment.
The art of maintaining cell or tissue cultures as well as microbial systems for studying genetics and cell physiology is well established. Means and methods are available for maintaining permanent cell lines, prepared by successive serial transfers from isolated cells. For use in research, such cell lines are maintained on a solid support in liquid medium, or by growth in suspension containing support nutriments. Scale-up for large preparations seems to pose only mechanical problems.